The hypothesis of this renewal grant is that, on binding to estrogen receptors (i.e., ERa and ER(3) on the plasma membranes, estrogen rapidly activates receptor subtype-specific intracellular signaling pathways (i.e., G-proteins) leading to endothelial nitric oxide (NO) synthase (eNOS) and extracellular signal-regulated kinases (ERK2/1) activation. Translocation of activated ERK2/1 stimulates the AP-1 (Jun/Fos dimers) transcription factors in the nucleus where they interact with ERa and ERp to reciprocally regulate eNOS and caveolin-1 expression by estrogen. Five specific aims will be studied by using the well-defined sheep uterine artery endothelial cell culture model and human umbilical cord vein endothelial cells. Aim 1: to determine the proximal membrane ER signaling events (G-protein activation) and if ERa and ERb play different roles in G-protein activation and eNOS-NO production and ERK2/1 pathway on estrogen stimulation. Aim 2: to determine if membrane and nuclear ERa and ERb are both involved in the reciprocal regulation of eNOS and caveolin-1 mRNA and protein expression by estrogen. Aim 3: to determine the role of c-Jun/Jun-B AP-1 in regulating eNOS and caveolin-1 expression by estrogen. Aim 4: to determine the transcriptional mechanism(s) underlying estrogen stimulation of endothelial eNOS expression, i.e., activation of eNOS promoter via ERa and ERb interaction with AP-1. Aim 5: to determine the mechanisms underlying down-regulation of endothelial caveolin-1 expression by estrogen, i.e., regulation of caveolin-1 promoter via ERa/ERb interaction with AP-1 and DNA methylation. These studies are of critical importance biologically because they are the first designed specifically for a comprehensive understanding of membrane ER initiated signaling cross-talk with classical nuclear ER via AP-1 to the regulation of eNOS and caveoln-1 gene activation in relation to endothelial NO production by estrogen unique to steroid receptor and endothelial biology. These studies are clinically important as dramatic estrogen biosynthesis and rise in uterine blood flow during pregnancy are essential for the bi-exchange between the mother and fetus and insufficient blood supply causes early embryonic loss, intrauterine growth restriction, preeclampsia, in utero fetal programming of adult diseases and reduced neonatal birthweight, which in turn inversely correlate to perinatal/neonatal morbidity/mortality and cardiovascular well-being of the mother. These studies are relevant to uncover the dilemma of the cardiovascular protective effects of estrogen.